The emerging field of checkpoint receptors has revealed a latent, and powerful, ability of the adaptive immune system to shrink or eliminate tumors. Single agent response rates for checkpoint inhibitors, such as anti-PD-1 are still relatively low and preliminary results suggest that combinatorial approaches are significantly more effective. Recent studies have demonstrated that co-expression of PD-1 and the emerging checkpoint molecule Tim-3 is frequent in tumor infiltrating lymphocytes (TIL) and pre-clinical data suggest that combined targeting of PD-1 and Tim-3 produces synergistic effects on tumor regression. However, it is not clear how antibodies targeting these proteins, either in mono- or duo-therapy, modulate downstream signaling events. Resistance (or therapeutic escape) could also occur due to compensatory upregulation of an alternative checkpoint receptor after blockade of a single receptor, mediated in part by intracellular signaling cross talk. Molecular or functional cooperation between these proteins has not been examined in the context of freshly isolated TIL from human cancer patients. Intriguingly, the PI3K/Akt/mTOR signaling pathway is known to be inhibited or activated by PD-1 or Tim-3, respectively, suggesting that this signaling pathway is an important node for the regulation of T cell exhaustion. Based on emerging data, we hypothesize that PD-1 and Tim-3 cooperate to control the function of tumor-infiltrating exhausted/effector CD8+ T cells, and that modulation of PD-1 and Tim-3 signaling cross-talk modulates T cell receptor (TCR) activation in cancer. We propose to define pathways of Tim-3/PD-1 signaling and functional crosstalk in tumor-infiltrating activated vs exhausted CD8+ CTL from head and neck cancer (HNC) patients. We will then determine the extent to which Tim-3 modifies the effects of PD-1 on activated effector vs. exhausted CTL in a mouse model of HNC. Lastly, we will define pathways of compensatory ICR expression and function in response to PD-1 or Tim-3 blockade.